CCBR-638

Maggie Cam

Nov 11, 2015

Background

We want to analyze two RNA-Seq datasets derived from total RNA of fractionated Protrusions (Ps) and cell body (CB) fractions of mouse fibroblast cells. We are interested in RNAs that are enriched in protrusions (i.e. have a high Ps/CB ratio) and want to identify subsets of these RNAs that are co-regulated by different factors.

The first dataset aims at identifying RNAs whose enrichment at protrusions is (or is not) affected by knockdown of the APC tumor suppressor protein. We have sequenced 4 control (si-control) and 4 APC knockdown (si-APC) replicates. Each replicate consists of paired Ps and CB fractions (i.e. 16 samples total).

The second dataset aims at identifying RNAs whose enrichment at protrusions is affected by expression of a competing UTR construct, which mislocalizes RNAs through sequestration of necessary factors. We have sequenced 4 control (HBB) and 4 experimental (Pkp4) replicates. Each replicate consists of paired Ps and CB fractions (i.e. 16 samples total).

Data Processing

## Bioconductor version 3.2 (BiocInstaller 1.20.0), ?biocLite for help

This part of the program was run on biowulf, and data transferred to a local directory

#Command line version
module load subread
x=$(ls *.bam)
featureCounts -p -T 8 -s 2 -p -t exon -g gene_id -a /data/maggiec/RNASeq/Genomes/mm10/gencode.vM4.all.gtf -o counts_ss.txt $x

#Used R version:
gtf="/data/maggiec/RNASeq/Genomes/mm10/gencode.vM4.all.gtf"
targets <- readTargets()

fc <- featureCounts(files=targets$bam,isGTFAnnotationFile=TRUE,nthreads=32,
      annot.ext=gtf,GTF.attrType="gene_name",strandSpecific=2,isPairedEnd=TRUE)
x <- DGEList(counts=fc$counts, genes=fc$annotation)

Load data from local directory:

## [1] "fc"      "gtf"     "targets" "x"
##                     bam Cell Compartment Replicate Phenotype
## 1   Sample_HBB_CB_1.bam  HBB          CB         1    HBB_CB
## 2   Sample_HBB_CB_2.bam  HBB          CB         2    HBB_CB
## 3   Sample_HBB_CB_3.bam  HBB          CB         3    HBB_CB
## 4   Sample_HBB_CB_4.bam  HBB          CB         4    HBB_CB
## 5   Sample_HBB_Ps_1.bam  HBB          Ps         1    HBB_Ps
## 6   Sample_HBB_Ps_2.bam  HBB          Ps         2    HBB_Ps
## 7   Sample_HBB_Ps_3.bam  HBB          Ps         3    HBB_Ps
## 8   Sample_HBB_Ps_4.bam  HBB          Ps         4    HBB_Ps
## 9  Sample_Pkp4_CB_1.bam Pkp4          CB         1   Pkp4_CB
## 10 Sample_Pkp4_CB_2.bam Pkp4          CB         2   Pkp4_CB
## 11 Sample_Pkp4_CB_3.bam Pkp4          CB         3   Pkp4_CB
## 12 Sample_Pkp4_CB_4.bam Pkp4          CB         4   Pkp4_CB
## 13 Sample_Pkp4_Ps_1.bam Pkp4          Ps         1   Pkp4_Ps
## 14 Sample_Pkp4_Ps_2.bam Pkp4          Ps         2   Pkp4_Ps
## 15 Sample_Pkp4_Ps_3.bam Pkp4          Ps         3   Pkp4_Ps
## 16 Sample_Pkp4_Ps_4.bam Pkp4          Ps         4   Pkp4_Ps
##                        Status Sample_HBB_CB_1.bam Sample_HBB_CB_2.bam
## 1                    Assigned            28219262            26002638
## 2        Unassigned_Ambiguity              440881              397555
## 3     Unassigned_MultiMapping             5851372             5156921
## 4       Unassigned_NoFeatures             1256514             1126695
## 5         Unassigned_Unmapped                   0                   0
## 6   Unassigned_MappingQuality                   0                   0
## 7  Unassigned_FragementLength                   0                   0
## 8          Unassigned_Chimera                   0                   0
## 9        Unassigned_Secondary                   0                   0
## 10     Unassigned_Nonjunction                   0                   0
## 11       Unassigned_Duplicate                   0                   0
##    Sample_HBB_CB_3.bam Sample_HBB_CB_4.bam Sample_HBB_Ps_1.bam
## 1             31276043            20215388            24174225
## 2               498844              336763              484750
## 3              6530007             4027521             8503721
## 4              1409443              802542              669031
## 5                    0                   0                   0
## 6                    0                   0                   0
## 7                    0                   0                   0
## 8                    0                   0                   0
## 9                    0                   0                   0
## 10                   0                   0                   0
## 11                   0                   0                   0
##    Sample_HBB_Ps_2.bam Sample_HBB_Ps_3.bam Sample_HBB_Ps_4.bam
## 1             21033948            21092274            21707668
## 2               409691              436089              417646
## 3              7014376             8130277             6366255
## 4               640754              529615              574098
## 5                    0                   0                   0
## 6                    0                   0                   0
## 7                    0                   0                   0
## 8                    0                   0                   0
## 9                    0                   0                   0
## 10                   0                   0                   0
## 11                   0                   0                   0
##    Sample_Pkp4_CB_1.bam Sample_Pkp4_CB_2.bam Sample_Pkp4_CB_3.bam
## 1              27137705             24478990             26951417
## 2                458180               380978               426721
## 3               6335410              5034420              5581745
## 4                996922               924351              1112709
## 5                     0                    0                    0
## 6                     0                    0                    0
## 7                     0                    0                    0
## 8                     0                    0                    0
## 9                     0                    0                    0
## 10                    0                    0                    0
## 11                    0                    0                    0
##    Sample_Pkp4_CB_4.bam Sample_Pkp4_Ps_1.bam Sample_Pkp4_Ps_2.bam
## 1              21872625             25005064             18667911
## 2                390419               499736               387524
## 3               5023467              9049572              6576287
## 4                754598               597725               551206
## 5                     0                    0                    0
## 6                     0                    0                    0
## 7                     0                    0                    0
## 8                     0                    0                    0
## 9                     0                    0                    0
## 10                    0                    0                    0
## 11                    0                    0                    0
##    Sample_Pkp4_Ps_3.bam Sample_Pkp4_Ps_4.bam
## 1              23950416             20046535
## 2                551285               386252
## 3               8631717              5899079
## 4                548449               513661
## 5                     0                    0
## 6                     0                    0
## 7                     0                    0
## 8                     0                    0
## 9                     0                    0
## 10                    0                    0
## 11                    0                    0

Mapping Rate:

QC Check: Look at raw signal distribution and median expression levels

Data is normalized by TMM: filtered number of genes

## [1] 12900    16

Run Voom

## null device 
##           1

After Normalization

## Using  as id variables

## null device 
##           1

unnamed_chunk_7snapshot
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Statistical Analysis of Experimental groups (lmFit)

##    celltypeHBB_CB celltypeHBB_Ps celltypePkp4_CB celltypePkp4_Ps
## 1               1              0               0               0
## 2               1              0               0               0
## 3               1              0               0               0
## 4               1              0               0               0
## 5               0              1               0               0
## 6               0              1               0               0
## 7               0              1               0               0
## 8               0              1               0               0
## 9               0              0               1               0
## 10              0              0               1               0
## 11              0              0               1               0
## 12              0              0               1               0
## 13              0              0               0               1
## 14              0              0               0               1
## 15              0              0               0               1
## 16              0              0               0               1
## attr(,"assign")
## [1] 1 1 1 1
## attr(,"contrasts")
## attr(,"contrasts")$celltype
## [1] "contr.treatment"
## [1] "HBB_CB"  "HBB_Ps"  "Pkp4_CB" "Pkp4_Ps"

Heatmap: Top genes

Provenance:

## R version 3.2.1 (2015-06-18)
## Platform: x86_64-apple-darwin13.4.0 (64-bit)
## Running under: OS X 10.10.4 (Yosemite)
## 
## locale:
## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
##  [1] reshape2_1.4.1       d3heatmap_0.6.1      reshape_0.8.5       
##  [4] knitr_1.11           rgl_0.95.1367        ggplot2_1.0.1       
##  [7] edgeR_3.12.0         limma_3.26.1         Rsubread_1.20.1     
## [10] BiocInstaller_1.20.0
## 
## loaded via a namespace (and not attached):
##  [1] Rcpp_0.12.1        magrittr_1.5       MASS_7.3-44       
##  [4] munsell_0.4.2      colorspace_1.2-6   stringr_1.0.0     
##  [7] plyr_1.8.3         tools_3.2.1        DT_0.1            
## [10] grid_3.2.1         gtable_0.1.2       png_0.1-7         
## [13] htmltools_0.2.6    yaml_2.1.13        digest_0.6.8      
## [16] RColorBrewer_1.1-2 formatR_1.2.1      base64enc_0.1-3   
## [19] htmlwidgets_0.5    evaluate_0.8       rmarkdown_0.8.1   
## [22] labeling_0.3       stringi_0.5-5      scales_0.3.0      
## [25] jsonlite_0.9.17    proto_0.3-10